African Queens find mates when males are rare

Abstract In butterflies and moths, male‐killing endosymbionts are transmitted from infected females via their eggs, and the male progeny then perish. This means that successful transmission of the parasite relies on the successful mating of the host. Paradoxically, at the population level, parasite transmission also reduces the number of adult males present in the final population for infected females to mate with. Here we investigate if successful female mating when males are rare is indeed a likely rate‐limiting step in the transmission of male‐killing Spiroplasma in the African Monarch, Danaus chrysippus. In Lepidoptera, successful pairings are hallmarked by the transfer of a sperm‐containing spermatophore from the male to the female during copulation. Conveniently, this spermatophore remains detectable within the female upon dissection, and thus, spermatophore counts can be used to assess the frequency of successful mating in the field. We used such spermatophore counts to examine if altered sex ratios in the D. chrysippus do indeed affect female mating success. We examined two different field sites in East Africa where males were often rare. Surprisingly, mated females carried an average of 1.5 spermatophores each, regardless of male frequency, and importantly, only 10–20% remained unmated. This suggests that infected females will still be able to mate in the face of either Spiroplasma‐mediated male killing and/or fluctuations in adult sex ratio over the wet–dry season cycle. These observations may begin to explain how the male‐killing mollicute can still be successfully transmitted in a population where males are rare.

tion, as well as potential differences in the behavior of males and females (Ancona et al., 2017). Despite the difficulty in estimating OSRs in the field, in butterflies we can accurately determine the frequency of successful matings via the detection of spermatophores in mated females. During mating, male butterflies transfer a spermatophore, containing both sperm and accessory factors, to the receptive female (Cardoso & Gilbert, 2007;Sanchez & Cordero, 2014).
As the chitinous neck of the spermatophore remains in place after copulation, the presence or absence of one or more spermatophores in the bursa copulatrix of females can be used to determine the frequency of successful mating (or at least successful spermatophore transfer) in the field (Burns, 1968). In Lepidoptera, the delivery of sperm from the spermatheca is further complicated by the presence of both functional sperm (eupyrene) and parasperm (apyrene sperm) whose role in Monarch butterflies is unclear. Parasperm could play a role in facilitating the success of the eusperm, provisioning the female or zygote and/or mediating postcopulatory sexual selection (Wedell et al., 2009;Whittington et al., 2017). However, the ratio of functional sperm to parasperm is not known in our study species, and we therefore simply use the presence of one or more spermatophores in field-collected females to document the observed levels of successful mating (the frequency of females carrying one or more spermatophores) and the apparent frequency of multiple mating (the number of spermatophores found in females that have re-mated, where each spermatophore represents mating with one male).
Previous observations in butterfly populations with a high frequency of maternally inherited male-killing endosymbionts, such as Wolbachia or Spiroplasma, show that a scarity of males can indeed increase the percentage of virgins found in a field population. For instance, 94% of female Acrea encedon, infected with a male-killing Wolbachia, remained as virgins in a Ugandan population (Jiggins et al., 2000). Similarly, 50% of female Hypolimnas bolina in Samoa, infected with a different male-killing Wolbachia, remained unmated (Dyson & Hurst, 2004). Male killing can, in theory, benefit the parasitic endosymbiont through increased resource availability to infected female offspring (in the absence of their brothers), thereby increasing the transmission to future generations (Hurst et al., 1993). However, a failure of infected females to find mates, when sex ratios are strongly female-biased, could also impose a cost of the male-killing phenotype on the endosymbiont itself. Despite this potential cost to the male killer, infected Samoan populations of H. bolina have persisted for over 100 years despite a highly female-biased sex ratio of 100:1 (Dyson & Hurst, 2004). In fact, this apparently stable situation has only recently been disrupted by the emergence of host resistance to male killing on the island (Hornett et al., 2014;Reynolds et al., 2019). The suggested presence of this fitness cost to the male-killing microbe, in the form of unmated females that fail to transmit the male killer, therefore makes untested assumptions about the relative efficiency of males and females in finding mates in the face of female-biased sex ratios.
Danaus chrysippus is found throughout Africa as a series of different color morphs or subspecies. These color morphs converge on East Africa where admixture polymorphism, driven by the winds surrounding the Inter-Tropical Convergence Zone (ITCZ), creates populations with all color morphs present. This zone of admixture has been termed a contact zone for the different subspecies, and within that contact zone, some individuals are infected with a male-killling mollicute called Spiroplasma (Smith et al., 2019). Previous work has shown that Spiroplasma infection is linked to the occurrence of a neo-W sex chromosome and associated changes in color pattern, and that both the endosymbiont and the neo-W are strictly maternally inherited (Martin et al., 2020;Smith et al., 2019). There therefore appears to have been a single infection of the neo-W carrying lineage (Martin et al., 2020) and subsequent spread of this Spiroplasma-

neo-W complex across a region centered on southern Kenya in East
Africa (Smith et al., 2019). This zone of infection overlaps with the broader contact zone where the different color morphs of D. chrysippus meet and inter-breed (Liu et al., 2022;Smith et al., 2019). In some parts of this contact zone, sex ratios are highly female-biased (Smith et al., 2019), raising the possibility that the scarcity of males may result in a failure of females to mate. In other areas, both infection rates and sex ratios vary between the wet and dry seasons (Hassan et al., 2013;Herren et al., 2007), possibly associated with seasonal migration of the different color morphs (Smith et al., 1997).
Here, we therefore sampled two East African populations carrying differing frequencies of the male-killing mollicute Spiroplasma (Jiggins et al., 2000) and showing dramatic seasonal variation of OSR, in order to test if a lower proportion of males leads to a lower frequency of mated females associated with a failure of females to find mates. At the first site, Kitengala in Kenya, male killing is prevalent (Smith et al., 2019), whereas at the second site, Nyamata in Rwanda, Spiroplasma infection rates currently still appear low (Ndatiman et al., 2022). Strikingly, we find that mating rates are unaffected by OSR variation at both sites, and that females still mate (receive one or more spermatophores) when males are rare. While the relative importance of male killing and sex-specific dispersal in these two populations remains unresolved, the fact that infected females can still find male mates suggests that there may currently be little localized fitness cost of male killing to the Spiroplasma endosymbiont itself. Together, these findings help explain how the male killer can persist in highly female-biased populations.

| Study sites and sampling
Butterflies were collected at two different field sites in East Africa, both within the D. chrysippus contact zone where the different color morphs fly together. The first, Kitengala, near Nairobi in Kenya, is known to harbor high frequencies of the male-killing Spiroplasma and to consistently display female-biased sex ratios with a mean of 74.5% females (Smith et al., 2019). The second site, Nyamata in Rwanda, is highly polymorphic for butterfly color pattern while infection rates still appear low (Ndatiman et al., 2022). Sampling at Nyamata was carried out throughout 2021 only (N = 787), while at Kitengela, butterflies (N = 304) were collected between May 2013 and July 2014.
Adult males and females were collected using a butterfly net and a random subset of females (all females if <10 collected per month or 10 randomly selected females if more than 10 were collected per month) was selected for dissection (Nyamata dissected N = 102 and Kitengela N = 250). A successful mating was defined as the presence of one or more spermatophores in a single dissected female. Mating rates were defined as the number of spermatophores found in mated females (where each spermatophore is inferred to come from a mating with a different male).

| Statistical analyses
Adult sex ratio, or OSR, was calculated as the percentage of males in the sample population for each month and site (Figure 1a,b). To examine the possible effect of OSR on the frequency of successful matings, we ran two different generalized linear models (GLMs), using the 'glm2' function in the R Statistical Software (v4 1.2; R Core F I G U R E 1 Number of butterflies collected, number of females examined for spermatophores, and percentage of males in the sample population by month and study site: (a) Kitengela, Kenya, and (b) Nyamata, Rwanda; *Indicating months excluded from data analysis due to small sample size. included study site as a predictor variable. We also tested the interaction terms between location and monthly percentage of collected males, which were not significant in either model, and were therefore excluded from the final model itself. Months in which <10 butterflies were collected at either site, or less than six females dissected for spermatophores, were excluded from the analysis (Figure 1a,b for numbers of butterflies used in the analysis). We checked the Poisson model for under or over-dispersion, but no issues were detected.

| RE SULTS
Monthly adult sex ratio, presented as the percentage of males collected (Figure 1a

| DISCUSS ION
We wanted to test if the apparent scarcity of males in East African populations of the African Monarch can reduce mating success in female butterflies. Here, despite sampling limitations, we have shown that females at two different sites can still find mates even when males are rare. Here, we define rare as a low proportion of captured males relative to captured females. While we are currently unable to F I G U R E 2 Jitter plot showing the distribution of mated females (jittered around 1) and unmated females (jittered around 0) by monthly percentage of collected males and study location. Lines indicate fitted logistic regression curve by location. This may help explain why the current neoW-Spiroplasma complex appears to be spreading in East Africa, despite the fact that males are rare in some infected populations and at specific times in the wet-dry season cycle.
In the interpretation of our results, we hypothesize that OSRs differ in both time and space due to both intrinsic (e.g., male-killing) and extrinsic (e.g., sex-specific migration) factors and also due to likely behavioral differences in the apparency of males and females.
Thus, while we cannot validate the precise accuracy of our OSR estimates, and while the periods sampled differ at each site, we have convincingly shown that males are rare at both sites at certain times of the wet-dry season cycle. Strikingly, despite the apparent scarcity of males at some times of year, the number of spermatophores per female (mean of ~1.5 per female) remains remarkably constant.
There is therefore no clear relationship between female mating success and sex ratio at either site sampled. Without precise data on the relative roles of male killing (PCR determined rates of Spiroplasma infection) and sex-specific migration (differential movement of males and females in and out of the study sites), it is difficult to precisely interpret the small differences in mating frequencies observed between the two sites. Thus, we cannot determine if females need to mate multiply to achieve full fecundity (Wiklund et al., 2001) or if female choice plays a role in driving the different mating rates observed. However, we do note that the higher mating rate in Kenyan butterflies is consistent with theory that suggests that polyandry may evolve to overcome issues with sperm limitation (Wedell, 2013).
In turn, the Rwandan population was often male-biased, with mating rates still remaining low, and this is not consistent with the idea that male harassment drives mating rates (Andersson et al., 2000). These complications aside, however, our finding that only 11% of females remain unmated in the face of extensive male killing by Spiroplasma at Kitengela, Kenya, contrasts markedly with the 50-90% of unmated females found in other male-killer-infected species (Dyson & Hurst, 2004;Jiggins et al., 2000). Taken together, our results therefore suggest that female African Queens can still find mates even when males appear rare, for whatever extrinsic (ecological) or intrinsic (male killing) reason.
To fully understand the likely effects of female mating success on male-killer fitness, we need to take our previous findings into account. In brief, previous work has uncovered the recent emergence (~2200 years ago) of a single Spiroplasma infection, linked to a single origin of a new female-limited sex chromosome, here termed simply the neo-W (Martin et al., 2020;Smith et al., 2019). The neo-W and Spiroplasma are maternally co-inherited, forming a neo-W-Spiroplasma 'complex' that shows a surprisingly high frequency in the eastern parts of the hybrid zone between the different D. chrysippus subspecies (Martin et al., 2020). We expect that the highly female-biased sex ratios generated by the neo-W-Spiroplasma complex should result in an increase in the proportion of unmated female butterflies, as discussed above. As Spiroplasma is transmitted from mated females to their infected eggs, the presence of excess virgin females in a population should theoretically lead to a decline in the infected female population and potentially also limit the rate at which male killing can spread. This prediction, however, is based on the assumption that F I G U R E 3 Relationship between number of spermatophores and the proportion of males in monthly samples at both study locations. The size of circles and triangles depicts the number of females in each monthly sample. Lines indicate fitted regression curve by location, and gray shading indicates 95% confidence interval.